Importin ß1 targeting by hepatitis C virus NS3/4A protein restricts IRF3 and NF-κB signaling of IFNB1 antiviral response.

In this study, newly identified host interactors of hepatitis C virus (HCV) proteins were assessed for a role in modulating the innate immune response. The analysis revealed enrichment for components of the nuclear transport machinery and the crucial interaction with NS3/4A protein in suppression of interferon-ß (IFNB1) induction. Using a comprehensive microscopy-based high-content screening approach combined to the gene silencing of nuclear transport factors, we showed that NS3/4A-interacting proteins control the nucleocytoplasmic trafficking of IFN regulatory factor 3 (IRF3) and NF-κB p65 upon Sendai virus (SeV) infection. Notably, importin ß1 (IMPß1) knockdown-a hub protein highly targeted by several viruses-decreases the nuclear translocation of both transcription factors and prevents IFNB1 and IFIT1 induction, correlating with a rapid increased of viral proteins and virus-mediated apoptosis. Here we show that NS3/4A triggers the cleavage of IMPß1 and inhibits nuclear transport to disrupt IFNB1 production. Importantly, mutated IMPß1 resistant to cleavage completely restores signaling, similar to the treatment with BILN 2061 protease inhibitor, correlating with the disappearance of cleavage products. Overall, the data indicate that HCV NS3/4A targeting of IMPß1 and related modulators of IRF3 and NF-κB nuclear transport constitute an important innate immune subversion strategy and inspire new avenues for broad-spectrum antiviral therapies.

Elucidating novel hepatitis C virus-host interactions using combined mass spectrometry and functional genomics approaches.

More than 170 million people worldwide are infected with the hepatitis C virus (HCV), for which future therapies are expected to rely upon a combination of oral antivirals. For a rapidly evolving virus like HCV, host-targeting antivirals are an attractive option. To decipher the role of novel HCV-host interactions, we used a proteomics approach combining immunoprecipitation of viral-host protein complexes coupled to mass spectrometry identification and functional genomics RNA interference screening of HCV partners. Here, we report the proteomics analyses of protein complexes associated with Core, NS2, NS3/4A, NS4B, NS5A, and NS5B proteins. We identified a stringent set of 98 human proteins interacting specifically with one of the viral proteins. The overlap with previous virus-host interaction studies demonstrates 24.5% shared HCV interactors overall (24/98), illustrating the reliability of the approach. The identified human proteins show enriched Gene Ontology terms associated with the endoplasmic reticulum, transport proteins with a major contribution of NS3/4A interactors, and transmembrane proteins for Core interactors. The interaction network emphasizes a high degree distribution, a high betweenness distribution, and high interconnectivity of targeted human proteins, in agreement with previous virus-host interactome studies. The set of HCV interactors also shows extensive enrichment for known targets of other viruses. The combined proteomic and gene silencing study revealed strong enrichment in modulators of HCV RNA replication, with the identification of 11 novel cofactors among our set of specific HCV partners. Finally, we report a novel immune evasion mechanism of NS3/4A protein based on its ability to affect nucleocytoplasmic transport of type I interferon-mediated signal transducer and activator of transcription 1 nuclear translocation. The study revealed highly stringent association between HCV interactors and their functional contribution to the viral replication cycle and pathogenesis.